Blood is a major tissue of the human body, and has a predominant role the delivery of oxygen from the lungs to peripheral tissues. This role is carried out by erythrocytes, i.e., red blood cells (RBC). The oxygen is furnished to the peripheral cells from the lungs by an exchange-diffusion system brought about by a red, iron-containing protein called hemoglobin. When hemoglobin combines with oxygen, oxyhemoglobin is formed and when oxygen is given up to the tissues, the oxyhemoglobin is reduced to deoxyhemoglobin.
The red cell membrane is composed of two major structural units, the membrane bilayer and a cytoskeleton. A lipid bilayer and integral membrane proteins form the membrane bilayer, which has little structural strength and fragments readily by vesiculation. The other major component, the membrane skeleton, stabilizes the membrane bilayer and provides resistance to deformation. The cytoskeleton is linked to the bilayer in the erythrocyte membrane, possibly by lipid-protein as well as protein-protein associations. The hemoglobin, and other RBC components, are contained within the red cell membrane.
In adults, bone marrow is active in the formation of new red blood cells. Once erythrocytes enter the blood, these cells have an average lifetime of about 120 days. In an average person, about 0.83% of the erythrocytes are destroyed each day by phagocytosis, hemolysis or mechanical damage in the body, and the depleted cells are renewed from the bone marrow.
A wide variety of injuries and medical procedures require the transfusion of whole blood or a variety of blood components. Every patient does not require whole blood and, in fact, the presence of all of the blood components can cause medical problems. Separate blood fractions, such as erythrocytes and platelets, can be stored under those special conditions best suited to assure their biological activity at the time of transfusion. For example, when donor blood is received at a processing center erythrocytes are separated and stored by various methods. Such cells are storable in citrate-phosphate-dextrose at 4.degree. C. for up to five weeks, generally as a unit of packed erythrocytes having a volume of from 200 to 300 ml and a hematocrit value (expressed as corpuscular volume percent) of 70 to 90. Erythrocytes may also be treated with glycerol and then frozen at from -30.degree. to -196.degree. C. and stored for up to seven years in a glycerol solution, but must be kept frozen at low temperatures in order to survive sufficiently for transfusion. Both these methods require careful maintenance of storage temperature to avoid disruption of the desired biological activity of the erythrocytes, and provide a twenty-four hour survival time for at least 70% of the transfused cells, which is considered to be an acceptable level for use in transfusion practice in accordance with the American Association of Blood Bank standards.
It has thus been a desideratum to obtain reconstitutable cells, particularly red blood cells and platelets, which can be stored at high storage temperatures (4.degree. C. to room temperatures) with good shelf life.
Prior to the present invention, it has been believed to be impossible to freeze-dry cells in a manner which permits the reconstitution with an intact cytoskeleton and, in the case of erythrocytes, with biologically-active hemoglobin, i.e., viable red blood cells. Viable RBC's can be characterized by one or more of the following: capability of synthesizing ATP; cell morphology; P.sub.50 values; oxy, met and heme values; MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival. When RBC's have been lyophilized according to previous methods, for example in either an aqueous or phosphate-buffered saline (PBS) solution, the reconstituted cells are damaged to the extent that the cells are not capable of metabolizing or synthesizing ATP, and the cell hemoglobin cannot transport oxygen. Glutaraldehyde-fixed erythrocytes, which have been lyophilized and reconstituted, have found use primarily in agglutination assays.
Platelets are single cells in the circulation involved in the process of hemostasis. They are involved directly in the coagulation process. In cases where damage to vascular tissue occurs, platelets act by adherence to collagen and basement membranes which have been exposed. Following adherence, there is a release of constituents from intracellular granules. These compounds promote vasoconstriction, and aggregation of other platelets in the area of damage. The consequences of this behavior are an arresting of bleeding in damaged blood vessels and stimulation of coagulation.
Platelets consist of fragments of megakaryocyte mother cells. They range from 5 .mu.m.sup.3 to 12 .mu.m.sup.3 in size with an average of 7.1-7.5 .mu.m.sup.3. They are shaped like discs with numerous invaginations in the membrane. It is covered by a protein coat which is involved in the activation of coagulation. The membrane is composed of phospholipids, beneath which are submembranous filaments of actomyosin.
Platelets are formed from megakaryocytes in the bone marrow. They enter the circulation by fragmentation of the megakaryocyte. They survive in the circulation for about 10 days. Most remain in the general circulation, but about one third remain as a pool in the spleen.
A variety of injuries calls for the transfusion of platelets. Most involve cases in which bleeding is excessive. Platelets are generally storable after separation from whole blood which had been drawn into citrate-dextrose-phosphate-adenine (CPDA). This separation must normally be performed within six hours of collection with the blood at room temperature (22.degree.). The platelets are normally stored as concentrates in containers composed of polyolefin for periods up to 5-7 days at room temperature. The risks of bacterial growth in solutions stored at room temperature for this period limits the time during which platelets may be used in transfusion medicine to five days, as established by the FDA. Storage in liquid form at temperatures below room temperature leads to substantial loss in platelet functions such as platelet aggregation and release responses, membrane glycoprotein expression, etc. Solutions such as DMSO devised for freezing platelets pose problems due to toxicity and the ability of the platelets to withstand freezing.
It has also been a desideratum, therefore, to obtain cells, particularly platelets, which could be stored for prolonged periods of time at high storage temperatures (4.degree. C. to RT). Prior to the present invention, it has been believed impossible to freeze-dry cells, including platelets, in a manner which permits their reconstitution with intact membranes, functional enzymes, and preserved aggregation, release, and phagocytosis responses, i.e., viable platelets. Viable platelets can be characterized by one or more of the preceding characteristics. When platelets have been lyophilized according to previous methods, for example in PBS, the reconstituted cells do not have intact membranes, do not exhibit normal morphologies, are not capable of aggregating upon stimulating with ADP, and do not exhibit normal phagocytosis.